Heterogeneous Intracellular Localization and Expression of Ataxin-3

doi:10.1006/nbdi.1998.0208

Neurobiology of Disease

Volume 5, Issue 5, November 1998, Pages 335-347

https://doi.org/10.1006/nbdi.1998.0208 Get rights and content

Abstract

Spinocerebellar ataxia type 3 or Machado–Joseph disease (SCA3/MJD) is an autosomal dominant neurodegenerative disorder caused by an unstable and expanded CAG trinucleotide repeat that leads to the expansion of a polyglutamine tract in a protein of unknown function, ataxin-3. We have generated and characterized a panel of monoclonal and polyclonal antibodies raised against ataxin-3 and used them to analyze its expression and localization. In Hela cells, multiple isoforms are expressed besides the major 55-kDa form. While the majority of ataxin-3 is cytosolic, both immunocytofluorescence and subcellular fractionation studies indicate the presence of ataxin-3, in particular, of some of the minor isoforms, in the nuclear and mitochodrial compartments. We also show that ataxin-3 can be phosphorylated. In the brain, only one ataxin-3 isoform containing the polyglutamine stretch was detected, and normal and mutated proteins were found equally expressed in all patient brain regions analyzed. In most neurons, ataxin-3 had a cytoplasmic, dendritic, and axonal localization. Some neurons presented an additional nuclear localization. Ataxin-3 is widely expressed throughout the brain, with a variable intensity specific for subpopulations of neurons. Its expression is, however, not restricted to regions that show intranuclear inclusions and neurodegeneration in SCA3/MJD.

References (45)

M.W. Becher et al.
Intranuclear neuronal inclusions in Huntington's disease and dentatorubral and pallidoluysian atrophy: Correlation between the density of inclusions andIT15
Neurobiol. Dis.
(1998)
S.W. Davies et al.
Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation
Cell
(1997)
M. Difiglia et al.
Huntingtin is a cytoplasmic protein associated with vesicules in human and rat brain neurons
Neuron
(1995)
S. Hunot et al.
Nitric oxide synthetase and neuronal vulnerability in Parkinson's disease
Neuroscience
(1996)
H.L. Paulson et al.
Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3
Neuron
(1997)
C. Rochette-Egly et al.
Stimulation of RAR alpha activation function AF-1 through binding to the general transcription factor TFIIH and phosphorylation by CDK7
Cell
(1997)
C. Ross
When more is less: Pathogenesis of glutamine repeat neurodegenerative diseases
Neuron
(1995)
E. Scherzinger et al.
Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo
Cell
(1997)
A.H. Sharp et al.
Widespread expression of Huntington's disease gene (IT15) protein product
Neuron
(1995)
G. Stevanin et al.
Linkage disequilibrium between the spinocerebellar ataxia 3/Machado–Joseph disease mutation and two intragenic polymorphisms, one of which, X359Y, affects the stop codon
Am. J. Hum. Genet.
(1997)

G. Wang et al.

Machado–Joseph disease gene product identified in lymphocytes and brain

Biochem. Biophys. Res. Commun.

(1997)

C.L. Wellington et al.

Caspase cleavage of gene products associated with triplet expansion disorders generates trunccated fragments containing the polyglutamine tract

J. Biol. Chem.

(1998)

C. Brou et al.

Distinct TFIID complexes mediate the effect of different transcriptional activators

EMBO J.

(1994)

G. David et al.

Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion

Nature Genet.

(1997)

D. Devys et al.

The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation

Nature Genet.

(1993)

M. Difiglia et al.

Aggregation of Huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain

Science

(1997)

Y. Feng et al.

Fragile X mental retardation protein: Nucleocytoplasmic shuttling and association with somatodendritic ribosomes

J. Neurosci.

(1997)

H.P. Gerber et al.

Transcriptional activation modulated by homopolymeric glutamine and proline stretches

Science

(1994)

J. Goto et al.

cDNA cloning and polymorphism of the Machado–Joseph disease (MJD) gene

Am. Soc. Hum. Genet.

(1997)

A.M. Graybiel et al.

Differences in tyrosine hydroxylase-like immunoreactivity characterize the mesostriatal innervation of striosomes and extrastriosomal matrix at maturity

Proc. Natl. Acad. Sci. USA

(1987)

C.A. Gutekunst et al.

Identification and localization of huntingtin in brain and human lymphoblastoid cell lines with anti-fusion protein antibodies

Proc. Natl. Acad. Sci. USA

(1995)

M. Holmberg et al.

Spinocerebellar ataxia type 7 (SCA7): A neurodegenerative disorder with neuronal intranuclear inclusions

Hum. Mol. Genet.

(1998)

Cited by (89)

Development of an AAV-Based MicroRNA Gene Therapy to Treat Machado-Joseph Disease
2019, Molecular Therapy Methods and Clinical Development
Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is a progressive neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAG repeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulates within inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brain stem. In the current study, a non-allele-specific ATXN3 silencing approach was investigated using artificial microRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The miATXN3 candidates were screened in vitro based on their silencing efficacy on a luciferase (Luc) reporter co-expressing ATXN3. The three best miATXN3 candidates were further tested for target engagement and potential off-target activity in induced pluripotent stem cells (iPSCs) differentiated into frontal brain-like neurons and in a SCA3 knockin mouse model. Besides a strong reduction of ATXN3 mRNA and protein, small RNA sequencing revealed efficient guide strand processing without passenger strands being produced. We used different methods to predict alteration of off-target genes upon AAV5-miATXN3 treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in a large animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areas affected in SCA3 patients. These results proved a strong basis to move forward to investigate distribution, efficacy, and safety of AAV5-miATXN3 in large animals.
Physiological and pathophysiological characteristics of ataxin-3 isoforms
2019, Journal of Biological Chemistry
Citation Excerpt :
These discrepancies may be explained by the isoform applied, the use of tags, and the localization within the protein as well as specific differences in the experiments like translational inhibition and inhibitor concentrations (80–88). Ataxin-3 was reported to be located in different compartments of the cell (3, 4, 46). Moreover, the nucleus was previously reported to be an essential site for MJD pathology (29, 30).
Ataxin-3 is a deubiquitinating enzyme and the affected protein in the neurodegenerative disorder Machado–Joseph disease (MJD). The ATXN3 gene is alternatively spliced, resulting in protein isoforms that differ in the number of ubiquitin-interacting motifs. Additionally, nonsynonymous SNPs in ATXN3 cause amino acid changes in ataxin-3, and one of these polymorphisms introduces a premature stop codon in one isoform. Here, we examined the effects of different ataxin-3 isoforms and of the premature stop codon on ataxin-3’s physiological function and on main disease mechanisms. At the physiological level, we show that alternative splicing and the premature stop codon alter ataxin-3 stability and that ataxin-3 isoforms differ in their enzymatic deubiquitination activity, subcellular distribution, and interaction with other proteins. At the pathological level, we found that the expansion of the polyglutamine repeat leads to a stabilization of ataxin-3 and that ataxin-3 isoforms differ in their aggregation properties. Interestingly, we observed a functional interaction between normal and polyglutamine-expanded ATXN3 allelic variants. We found that interactions between different ATXN3 allelic variants modify the physiological and pathophysiological properties of ataxin-3. Our findings indicate that alternative splicing and interactions between different ataxin-3 isoforms affect not only major aspects of ataxin-3 function but also MJD pathogenesis. Our results stress the importance of considering isoforms of disease-causing proteins and their interplay with the normal allelic variant as disease modifiers in MJD and autosomal-dominantly inherited diseases in general.
Experimental and Clinical Strategies for Treating Spinocerebellar Ataxia Type 3
2018, Neuroscience
Citation Excerpt :
Atx-3, the affected protein, is a ubiquitously expressed and predominantly cytoplasmic protein. However, in certain cell types, atx-3 was also observed in both the nucleus and the cytoplasm (Paulson et al., 1997; Wang et al., 1997; Schmidt et al., 1998; Tait et al., 1998; Trottier et al., 1998; Pozzi et al., 2008; Macedo-Ribeiro et al., 2009; Reina et al., 2010). Atx-3 belongs to the Josephin family, which is one of five different families of deubiquitinating enzymes.
Spinocerebellar ataxia type 3 (SCA3), or Machado–Joseph disease (MJD), is an autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) tract in the ataxin-3 protein. To date, there is no effective therapy available to prevent progression of this disease. However, clinical strategies for alleviating various symptoms are imperative to promote a better quality of life for SCA3/MJD patients. Furthermore, experimental therapeutic strategies, including gene silencing or mutant protein clearance, mutant polyQ protein modification, stabilizing the native protein conformation, rescue of cellular dysfunction and neuromodulation to slow the progression of SCA3/MJD, have been developed. In this study, based on the current knowledge, I detail the clinical and experimental therapeutic strategies for treating SCA3/MJD, paying particular attention to drug discovery.
Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function
2018, Neurochemistry International
Deubiquitinating enzymes (DUBs) play important roles in a variety of cellular processes, including regulation of protein homeostasis. The DUB ataxin-3 is an enzyme implicated in protein quality control mechanisms. In the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3), ataxin-3 contains an expanded polyglutamine (polyQ) stretch that leads to aggregation of the protein and neuronal dysfunction. Increasing the understanding of ataxin-3 protein interaction partners could help to elucidate disease mechanisms. Hence, we analyzed the repertoire of proteins interacting with normal and polyQ expanded ataxin-3 by mass spectrometry. This showed that both normal and polyQ expanded ataxin-3 interacted with components of the protein quality control system and mitochondria. Five proteins showed increased interaction with polyQ expanded ataxin-3 relative to normal and three of these were mitochondrial proteins. The analyses underline the role of ataxin-3 in ubiquitin biology and point towards a role in mitochondrial biology.
Toward understanding Machado-Joseph disease
2012, Progress in Neurobiology
Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is the most common inherited spinocerebellar ataxia and one of many polyglutamine neurodegenerative diseases. In MJD, a CAG repeat expansion encodes an abnormally long polyglutamine (polyQ) tract in the disease protein, ATXN3. Here we review MJD, focusing primarily on the function and dysfunction of ATXN3 and on advances toward potential therapies. ATXN3 is a deubiquitinating enzyme (DUB) whose highly specialized properties suggest that it participates in ubiquitin-dependent proteostasis. By virtue of its interactions with VCP, various ubiquitin ligases and other ubiquitin-linked proteins, ATXN3 may help regulate the stability or activity of many proteins in diverse cellular pathways implicated in proteotoxic stress response, aging, and cell differentiation. Expansion of the polyQ tract in ATXN3 is thought to promote an altered conformation in the protein, leading to changes in interactions with native partners and to the formation of insoluble aggregates. The development of a wide range of cellular and animal models of MJD has been crucial to the emerging understanding of ATXN3 dysfunction upon polyQ expansion. Despite many advances, however, the principal molecular mechanisms by which mutant ATXN3 elicits neurotoxicity remain elusive. In a chronic degenerative disease like MJD, it is conceivable that mutant ATXN3 triggers multiple, interconnected pathogenic cascades that precipitate cellular dysfunction and eventual cell death. A better understanding of these complex molecular mechanisms will be important as scientists and clinicians begin to focus on developing effective therapies for this incurable, fatal disorder.
Viral-based animal models in polyglutamine disorders
2024, Brain

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Regular ArticleHeterogeneous Intracellular Localization and Expression of Ataxin-3

Abstract

Neurobiol. Dis.

Cell

Neuron

Neuroscience

Neuron

Cell

Neuron

Cell

Neuron

Am. J. Hum. Genet.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Distinct TFIID complexes mediate the effect of different transcriptional activators

EMBO J.

Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion

Nature Genet.

The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation

Nature Genet.

Aggregation of Huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain

Science

Fragile X mental retardation protein: Nucleocytoplasmic shuttling and association with somatodendritic ribosomes

J. Neurosci.

Transcriptional activation modulated by homopolymeric glutamine and proline stretches

Science

cDNA cloning and polymorphism of the Machado–Joseph disease (MJD) gene

Am. Soc. Hum. Genet.

Differences in tyrosine hydroxylase-like immunoreactivity characterize the mesostriatal innervation of striosomes and extrastriosomal matrix at maturity

Proc. Natl. Acad. Sci. USA

Identification and localization of huntingtin in brain and human lymphoblastoid cell lines with anti-fusion protein antibodies

Proc. Natl. Acad. Sci. USA

Spinocerebellar ataxia type 7 (SCA7): A neurodegenerative disorder with neuronal intranuclear inclusions

Hum. Mol. Genet.

Regular Article
Heterogeneous Intracellular Localization and Expression of Ataxin-3